Inhibition of caspases promotes long-term survival and reinnervation by axotomized spinal motoneurons of denervated muscle in newborn rats

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Experimental Neurology 181 (2003) 190 –203

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Inhibition of caspases promotes long-term survival and reinnervation by axotomized spinal motoneurons of denervated muscle in newborn rats Yuen-Man Chan,a Leung-Wah Yick,a Henry K. Yip,a Kwok-Fai So,a Ronald W. Oppenheim,b and Wutian Wua,* a

b

Department of Anatomy, Faculty of Medicine, The University of Hong Kong, Hong Kong, China Department of Neurobiology and Anatomy and the Neuroscience Program, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA Received 13 September 2002; revised 10 December 2002; accepted 16 December 2002

Abstract We examined whether (1) a pan-caspase inhibitor, Boc-D-FMK, exerts long-term neuroprotective effects on spinal motoneurons (MNs) after root avulsion in neonatal rats and (2) whether the rescued spinal MNs regenerate their axons into a peripheral nerve (PN) graft and reinnervate a previously denervated target muscle. Eight weeks after root avulsion, 67% of spinal MNs remained in the Boc-D-FMK-treated group, whereas all MNs died in the sham control group. By 12 weeks postinjury, however, all Boc-D-FMK treated MNs died. In the regeneration experiment, a PN graft was implanted at different times after injury. The animals were allowed to survive for 4 weeks following the operation. Without caspase inhibition, MNs did not regenerate at any time point. In animals treated with Ac-DEVD-CHO, a caspase-3-specific inhibitor, and Boc-D-FMK, 44 and 62% of MNs, respectively, were found to regenerate their axons into a PN graft implanted immediately after root avulsion. When the PN graft was implanted 2 weeks after injury, however, MNs failed to regenerate following Ac-DEVD-CHO treatment, whereas 53% of MNs regenerated their axons into the graft after treatment with Boc-D-FMK. No regeneration was observed when a PN graft was implanted later than 2 weeks after injury. In the reinnervation study, injured MNs and the target biceps muscle were reconnected by a PN bridge implanted 2 weeks after root avulsion with administration of Boc-D-FMK. Eight weeks following the operation, 39% of MNs reinnervated the biceps muscle. Morphologically normal synapses and motor endplates were reformed in the muscle fibers. Collectively, these data provide evidence that injured neonatal motoneurons can survive and reinnervate peripheral muscle targets following inhibition of caspases. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Caspase inhibitor; Motoneurons; Long-term survival; Regeneration; Peripheral nerve bridging; Muscle reinnervation

Introduction Brachial plexus injury leads to massive MN degeneration. Since the target muscle lacks innervation, muscular atrophy occurs and therefore some patients lose part or all of their arm movement. Every year many infants suffer from this injury when they are delivered improperly (Hoeksma et al., 2000; Noetzel et al., 2001; Bar et al., 2001). A recent clinical strategy for the treatment of brachial plexus injury is to reimplant the avulsed root into the lesioned area (Carl* Corresponding author. Fax: ⫹852-2819-0857. E-mail address: [email protected] (W. Wu).

stedt et al., 2000; Fournier et al., 2001). However, surgical difficulties, determining an optimum site for reimplantation and the patients age all affect the outcome of recovery (Jamieson and Hughes, 1980; Fournier et al., 2001). Therefore, it is crucial to develop an effective treatment for pediatric brachial plexus injury. The age of the animal and the type of injury are two important factors that affect the fate of spinal MNs after axonal injury. In adult rats, the majority of spinal MNs survive following distal axotomy, whereas most of them die after root avulsion (Wu, 1993). In developing animals, by contrast, almost all MNs die after either distal axotomy or root avulsion (Yuan et al., 2000; Chan et al.,

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Y.-M. Chan et al. / Experimental Neurology 181 (2003) 190 –203

2001). Age also affects the regenerative capacity of spinal MNs after axonal injury. It has been shown that axonal regrowth into a PN graft is possible for adult CNS neurons (Sorbie and Porter, 1969; David and Aguayo, 1981, 1985; Richardson et al., 1982, 1984; Horvat, 1991; Emery et al., 1997) and following root avulsion in adult rats, spinal MNs are able to regenerate their axons into a reimplanted PN graft (Wu et al., 1994; Chai et al., 2000), whereas neonatal MNs are unable to regenerate into an implanted PN graft (Chan et al., 2002). The failure to regenerate in neonates may be due to (1) MN death before any regeneration occurs since virtually all spinal MNs degenerate within 7 days following root avulsion or (2) developing MNs may lose the capacity for regeneration after axonal injury. We have previously shown that caspase inhibitors increase the survival rate of neonatal spinal MNs for up to 3 weeks following root avulsion (Chan et al., 2001), which may provide a larger window for examining the regenerative capacity of injured developing MNs. It has been shown in adult animals that injured spinal MNs can regenerate through a PN bridge (Emery et al., 2000; Rhrich-Haddout et al., 2001) and form functional synaptic contacts (Horvat et al., 1989; Pecot-Dechavassine and Mira, 1994). The PN bridge consists of Schwann cells and extracellular molecules and both may play crucial roles in axonal regeneration (Villegas-Perez et al., 1988; Carbonetto, 1991; Morrissey et al., 1991; Guenard et al., 1992; Bunge, 1994). However, whether spinal MNs can reinnervate target muscles and form functional connections through a PN bridge in neonates remains unclear. Although the precise mechanism for MN death after axonal injury is not fully understood, many reports have shown that such death may be triggered by an apoptotic pathway (Rothstein et al., 1994; Yoshiyama et al., 1994; Lo et al., 1995; Oliveira et al., 1997; Li et al., 1998; Martin, 1999; Blondet et al., 2001; Liu and Martin, 2001a, 2001b). Caspases are important mediators of apoptosis (Milligan et al., 1995; Barnes et al., 1998; Hayashi et al., 1998; Turgeon et al., 1998) and inhibition of various kinds of caspases can significantly promote the survival of CNS neurons after injury. Peptide inhibitors of caspases arrest apoptotic cell death of MNs in both in vivo and in vitro models (Milligan et al., 1995; Allen et al., 2001; Gerhardt et al., 2001; Hayashi et al., 2001; von Coelln et al., 2001). We have previously found that a single dose of the pan-caspase inhibitor benzyloxycarbonyl-Asp(OMe)fluoromethylketone (BocD-FMK) rescued more than 70% of MNs for at least 3 weeks after root avulsion (Chan et al., 2001). Although the caspase-3-specific inhibitor N-acetyl-Asp-Glu-ValAsp aldehyde (Ac-DEVD-CHO) also promoted MN survival in this situation, the effect was lost by 2 weeks postinjury.

191

Materials and methods Surgical procedures On the day of birth, newborn female Spraque–Dawley rats were anesthetized under deep hypothermia. Under a surgical microscope, a dorsal laminectomy was carried out and the spinal root of the seventh cervical (C7) segment was identified. The C7 ventral root together with the dorsal root were avulsed by a pair of microhemostatic forceps. To study the long-term neuroprotective effect of Boc-DFMK, animals were divided into two groups. There were six rats in each group at each time point. The first group of animals received gel foam soaked with 10 ␮l of 0.01 M phosphate-buffered saline (PBS) placed onto the lesioned area to serve as a sham control. The second group of animals was treated with a piece of gel foam soaked with 0.5 ␮g/10 ␮l of Boc-D-FMK (Enzyme System Products, Livermore) placed at the lesioned site. Animals were allowed to survive for 8 or 12 weeks. To examine the regeneration-promoting effect of the caspase inhibitors, there were six rats in each group at each time point that received gel foam soaked with 10 ␮l of 0.01 M PBS (sham control), 0.5 ␮g/10 ␮l Boc-D-FMK, or 1 ␮g/10 ␮l Ac-DEVD-CHO (Bachem AG, Switzerland) placed into the lesioned area immediately after the injury. The PN graft was a 20-mm segment of the autologous sciatic nerve dissected immediately before implantation. The proximal end of the PN graft was placed and attached onto the surface of the spinal cord and fixed by 11-0 suture while the distal end of the graft was placed blindly outside the spinal cord. To investigate the optimum age for nerve implantation, the PN graft was implanted into the lesioned area at different time points after the injury. Since the neuroprotective effect of Ac-DEVD-CHO lasts for 2 weeks (Chan et al., 2001), the PN graft was either implanted immediately or 2 weeks after root avulsion. In the Boc-DFMK-treated animals, the PN graft was implanted either immediately or 2, 4, 6, or 8 weeks after root avulsion. Some animals received a second treatment with the caspase inhibitor during the implantation of the PN graft. Animals were allowed to survive for 4 weeks after the implantation of the PN graft. To examine the role of caspase inhibitors on MN regeneration, two sets of experiments were carried out. Our previous results suggested that the permissive period for MN regeneration began after the first 2 weeks of postnatal development (Chan et al., 2002). Whether caspase inhibitors directly promoted MNs regeneration could only be confirmed if caspase inhibitors act at ages earlier than 2 weeks. Therefore, in the first experiment, neonatal rats at days 1 (P1), 7 (P7), 14 (P14), 21 (P21), and 28 (P28) were used. Immediately after root avulsion, animals received PBS, Boc-D-FMK, or Ac-DEVD-CHO together with the implantation of a PN graft as described above. Six rats were used in each group and the animals were allowed to survive for

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4 weeks. In the second experiment, only neonatal day 1 (P1) rats were used. There were six animals in each group and they were treated with PBS, Boc-D-FMK, or Ac-DEVDCHO together with implantation of a PN graft immediately after root avulsion (as described above). The postoperative survival period was 10 days prior to when control animals first express a regenerative capacity (Chan et al., 2002). To investigate whether the regenerated axons could reinnervate their target, a 40-mm segment of the autologous sciatic nerve served as a PN bridge joining the motoneurons and biceps muscle. Since the sciatic nerve in neonates was not long enough to bridge the injured motoneurons and the biceps muscle, an autologous PN bridge was derived when animals were 2-week-old. Root avulsion was performed in neonatal day 1 rats and caspases inhibitors were placed immediately after the injury to keep the motoneurons alive before and after PN bridge implantation. Two groups of animals were used with six rats in each group. In the first group, animals received Boc-D-FMK (as described above) immediately after root avulsion and the gel foam soaked with Boc-DFMK remained in the lesioned area for 2 weeks until the PN bridge was implanted. Animals in the second group received PBS and PN bridge implantation and served as controls. For the PN bridge implantation, the proximal end of the graft was attached onto the surface of spinal cord while the distal end of the graft was inserted into the denervated biceps muscle. Possible reinnervation by the transected musculocutaneous nerve was prevented by removing a 10-mm segment of the nerve. A 11-0 suture was used to avoid dislocation of the graft from the biceps muscle and the spinal cord. Animals were allowed to survive for 8 weeks after the implantation of the PN graft. Retrograde labeling of motoneurons The regenerated MNs were identified by retrograde labeling with FluoroGold (FG, Flurochrome, 6% in distilled water). Two days before perfusion, 2 ␮l of FG was injected into the distal end of the nerve graft at a point 10 –15 mm from the spinal cord by using a microsyringe. For examining reinnervation, 2 ␮l of FG was injected into the biceps muscle. Perfusion and tissue processing At the end of the appropriate postoperative survival period, rats were deeply anesthetized with a lethal dose of ketamine and xylazine. They were perfused intracardinally with normal saline, followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The C7 spinal segment was removed and immersion-fixed for 6 h and then placed into 30% sucrose in 0.1 M PBS overnight.

␣-Bungarotoxin histochemistry To visualize the postsynaptic acetylcholine receptor (AChR), TRITC-conjugated ␣-bungarotoxin (␣-BTXTRITC, Molecular Probes, 10 ␮M) was used. Briefly, the muscle was sectioned (10 ␮m) with a cryostat. After blocking with 5% bovine serum albumin in 0.1 M PBS, the sections were incubated in ␣-BTX-TRITC for 2 h and then washed in PBS. The sections were examined under a fluorescent microscope (Nikon). Silver and cholinesterase staining of muscle reinnervation To visualize the nerve terminals and the motor endplates, fresh biceps muscle was frozen in liquid nitrogen and cut into 60 ␮m longitudinal sections. Following the protocol of Hopkins and Slack (1981), the sections were stained for cholinesterase in a solution containing 1.7 mM of acetylthiocholine iodide, 5 mM of sodium citrate, 30 mM of sodium maleate, 3 mM of cupric sulfate, and 0.5 mM of potassium ferricyanide (pH 6.0) at 4°C for 20 min. After rinsing in water, the sections were treated with 4 mM potassium ferricyanide for 10 min. Following two rinses in water, the sections were dehydrated in ascending concentration of ethanol and then rehydrated. The sections were fixed in 10% formalin. After treatment with pyridine at 37°C for 25 min, the sections were stained with 1% silver nitrate at 37°C for 40 min. Following four rinses in water, the sections were developed in a solution containing 90 mM hydroquinone and 400 mM sodium sulfite. After dehydration in ascending ethanol solutions, the sections were mounted and examined with a light microscope. Motoneuron counts and statistics Cryostat sections (30 ␮m) of spinal cord from the longterm survival group were stained with neutral red. The number of surviving motoneurons was counted on both the intact and the lesioned sides as described previously (Clarke and Oppenheim, 1995; Oppenheim et al, 1989; Wu, 1993; Wu and Li, 1993). The total number of surviving MNs on the lesioned side was expressed as a percentage of the number of motoneurons on the contralateral intact side. In the regeneration group, FG-positive cells were counted on the lesioned side under a fluorescent microscope (Nikon) and FG-labeled MNs were considered to have regenerated. After counting, serial sections were stained with neutral red and the number of surviving MNs was counted on the contralateral intact side. The total number of regenerated MNs was expressed as a percentage of FG-positive cells against the surviving cells on the contralateral intact side. The percentages of surviving or regenerating MNs were compared statistically among groups using one-way ANOVA followed by Tukey–Kramer multiple comparisons test. All data were expressed as mean ⫾ SEM.

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Table 1 Percentage survival and mean soma area (mean ⫾ SEM) of normal and avulsed motoneurons 8 weeks

%Surviving motoneurons Mean soma area (in ␮m2)

12 weeks

Normal

Avulsion

PBS

Boc-D-FMK

Normal

Avulsion

PBS

Boc-D-FMK

100 191.03⫾4.43

0 N/A

0 N/A

67.3 ⫾ 1.33 193.67 ⫾ 4.80 (P ⬎ 0.05)

100 204.34⫾5.18

0 N/A

0 N/A

0 N/A

Results Long-term neuroprotective effect of Boc-D-FMK Motoneurons were identified and counted as described previously (Clarke and Oppenheim, 1995). In brief, only MNs with a large nucleus containing clearly visible nucleoli and a largely distinct cytoplasm were counted. Because the number of MNs on the contralateral intact side of the experimental animals was not significantly different from normal control animals (data not shown), the contralateral side served as an internal control. We have previously reported that by 7 days postlesion, there are no surviving MNs on the lesioned side of untreated control animals (Chan et al., 2001). By 8 weeks after root avulsion, treatment with the general caspase inhibitor Boc-D-FMK resulted in the survival of 67.3 ⫾ 1.33% of injured MNs (Table 1). The rescued MNs showed normal morphological features characterized by large cell bodies, centrally localized nuclei, and clear Nissl staining (Fig. 1E). The average cell size of the surviving MNs (193.67 ⫾ 4.80 ␮m2) was similar to MNs on the intact side (191.03 ⫾ 4.43 ␮m2) (Table 1). By 12 weeks following root avulsion, all MNs on the lesioned side had died (Fig. 1F). These data indicate that the neuroprotective effect of Boc-D-FMK lasts for about 8 weeks.

8.47 ␮m2) (P ⬎ 0.05) (Table 2). Since FG was injected 10 –15 mm from the lesioned site, the MNs had regenerated their axons into the PN graft over a distance of at least 10 mm. Nonregenerated MNs (unlabeled) were slightly but not significantly smaller in mean soma size (168.22 ⫾ 2.79 ␮m2) compared to the contralateral intact MNs (176.77 ⫾ 8.47 ␮m2) (P ⬎ 0.05). When the implantation of the PN graft was delayed for 4, 6, or 8 weeks following root avulsion, no regenerated MNs were observed even when a second dose of Boc-D-FMK was applied. The cell soma size and the morphological features of the surviving MNs were similar to normal MNs (P ⬎ 0.05) (Table 3). Therefore, the optimum time for nerve implantation in neonatal animals was within 2 weeks after root avulsion. In the Ac-DEVD-CHO-treated group, 44.1 ⫾ 4.80% of MNs regenerated their axons into a PN graft transplanted into the lesioned site immediately following root avulsion (Fig. 2G). Ac-DEVD-CHO-treated MNs were unable to regenerate their axons into the implanted PN graft when the graft implantation was delayed for 2 weeks (Fig. 2H). Some lesioned MNs still remained on the lesioned side but their soma size (129.69 ⫾ 2.53 ␮m2) was markedly smaller than normal contralateral MNs (176.77 ⫾ 8.47 ␮m2; P ⬍ 0.001) (Table 2). Therefore, Ac-DEVD-CHO could only promote regeneration when the nerve graft was implanted immediately after root avulsion.

Regenerative-promoting effect of caspase inhibitors Effect of caspase inhibitors on regenerative capacity All animals were allowed to survive for 4 weeks after implantation of the PN graft. The percentage of FG-positive MNs was expressed as the number of FG-positive MNs compared to the number of MNs with clearly recognized nucleoli by neutral red staining on the contralateral side. After the implantation of a PN graft alone (or sham treatment), no MNs were labeled by FG on the lesioned side of C7 (Fig. 2A–D). Treatment with Boc-D-FMK resulted in 61.6 ⫾ 5.88% of MN regeneration when the PN graft was implanted immediately after root avulsion (Fig. 2E) and 52.5 ⫾ 1.65% when the PN graft was implanted 2 weeks postinjury (Fig. 2F and Table 2). Morphologically, the regenerating MNs appeared similar to the MNs present on the contralateral intact side (i.e., showing large cell bodies and clearly visualized nucleoli). The cell soma size of the regenerated MNs (193.93 ⫾ 5.46 ␮m2) was comparable to normal MNs (176.77 ⫾

In a previous study, we examined the regenerative capacity of axotomized spinal MNs during postnatal development (Chan et al., 2002) and observed that only after the first 2 weeks of postnatal development was MN regeneration possible. In the present study, we found that at P1 and P7, all MNs died by 4 weeks after root avulsion, despite the presence of the PN graft, and therefore no MNs regenerated their axons into the graft (Fig. 3A and B and Fig. 4A). In P14 rats, implantation of a PN graft resulted in 30.8 ⫾ 1.56% of the MNs regenerating axons (Fig. 3C and Fig. 4A), and an increasing number of MNs regrew their axons into the PN graft at older ages: 55.4 ⫾ 3.27 and 60.1 ⫾ 2.17% of regenerated motoneurons in P21 and P28 rats respectively (Fig. 3D and E and Fig. 4A). Therefore, only after the first 2 weeks of postnatal development can MNs exhibit regeneration after injury.

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Fig. 1. Photomicrographs of cross sections of C7 spinal cord either 8 or 12 weeks following root avulsion. The sections were stained with neutral red. Dashed line indicates the ventral horn of the spinal cord. (A and B) Motoneurons of normal animals 8 (A) or 12 (B) weeks of age. (C) Control animals treated with PBS 8 weeks after root avulsion. (D) Control animals treated with PBS 12 weeks after injury. Motoneurons are absent in the 8 and 12 week controls. (E) By 8 weeks postinjury, about 67% of injured motoneurons were rescued by Boc-D-FMK. The rescued motoneurons were morphologically similar to the normal motoneurons. (F) By 12 weeks MNs had degenerated following root avulsion in Boc-D-FMK-treated animals. Scale bar ⫽ 100 ␮m.

Treatment with Boc-D-FMK significantly enhanced the regenerative capacity in spinal MNs after root avulsion in developing animals. Treatment with Boc-D-FMK resulted in 61.6 ⫾ 2.40% regeneration on P1 (Fig. 3F and Fig. 4A), 61.5 ⫾ 2.36% on P7 (Fig. 3G and Fig. 4A), 60.8 ⫾ 2.58% on P14 (Fig. 3H and Fig. 4A), and 62.3 ⫾ 2.31 and 63.2 ⫾ 2.47% on P21 and P28 (Fig. 3I and J and Fig. 4A). Admin-

istration of Ac-DEVD-CHO was as effective as Boc-DFMK in promoting the regeneration of spinal MNs after root avulsion at different postnatal ages (Fig. 3K–O and Fig. 4A). During postnatal development, spinal MNs begin to express a regenerative capacity at around 2 weeks of age (Chan et al., 2002). P1 animals allowed to survive for 10

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Fig. 2. Photomicrographs of C7 spinal cord sections showing the regenerated MNs after treatment with Boc-D-FMK or Ac-DEVD-CHO at different time points. (A and B) MNs on the contralateral intact side were not labeled by FG, indicating that MNs in the contralateral intact side did not contribute to the regenerated axons in the PN graft. (C and D) Motoneurons on the lesioned sides were FG-negative when the PN graft was implanted immediately (C) or 2 weeks (D) after root avulsion without treatment of caspase inhibitors. (E) Retrograde labeling with FG showed that the administration of Boc-D-FMK enhanced regeneration when the PN graft was implanted immediately after the lesion. (F) Boc-D-FMK treated motoneurons could also regrow their axons into a PN graft implanted 2 weeks following root avulsion. (G) Regenerated motoneurons were also found in the Ac-DEVD-CHO-treated animals with immediate implantation of PN graft. (H) Motoneurons did not regenerate in the Ac-DEVD-CHO-treated group when the implantation of PN graft was delayed for 2 weeks. Scale bar ⫽ 100 ␮m.

days after the implantation of a PN graft had not yet developed a regenerative capacity and therefore the PN graft was ineffective (Fig. 4B). However, caspase inhibition together with the PN graft promoted the regeneration of P1 spinal

MNs (Fig. 4C and D). These results suggest that caspase inhibitors may directly promote regeneration rather than merely keep MNs alive until they are old enough (2 weeks) to express the capacity for regeneration.

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Table 2 Percentage of FG-positive MNs and mean soma area (mean ⫾ SEM) from normal and avulsed animals

%FG-positive motoneurons Mean soma area (in ␮m2) FG positive FG negative

PN graft implanted immediately after lesion

PN graft implanted 2 weeks after lesion

Normal

PBS

Normal

PBS

0

0

0

0

N/A

N/A

149.51 ⫾ 5.48

N/A

N/A

160.7 ⫾ 3.29

N/A

138.5 ⫾ 7.14 (P ⬎ 0.05)

176.77 ⫾ 8.47

N/A

Boc-D-FMK 61.6 ⫾ 5.88 154.39 ⫾ 2.94 (P ⬎ 0.05) 141.2 ⫾ 1.03 (P ⬎ 0.05)

Reinnervation of biceps muscles by regenerated motoneurons Since all injured MNs degenerated in the control group, no FG-labeled MNs were observed in these animals (Fig. 5A). By contrast, 38.8 ⫾ 1.02% FG-labeled MNs were observed in lesioned animals treated with Boc-D-FMK and a PN graft (Fig. 5B) indicating successful regeneration in this situation. Morphological studies of the reinnervated biceps We reasoned that successful reinnervation of the denervated biceps muscle had occurred if (1) the muscle did not show significant signs of atrophy and (2) morphologically normal neuromuscular junctions could be observed. In the intact muscle, multinucleated muscle fibers could be seen clearly in both cross (Fig. 6A) and longitudinal (Fig. 6B) sections. Muscle fibers were evenly distributed and striations could be observed in longitudinal sections. Nerve fibers innervated endplates (Fig. 5C), and in normal biceps muscle, single MN axons innervated each endplate. Eight weeks after denervation, the biceps muscle exhibited some necrosis (Fig. 6C and D), and nerve fibers did not innervate endplates (Fig. 5E). In the Boc-D-FMK-treated animals, muscle fibers with striations were found (Fig. 6E and F) although they were smaller than normal. Bundles of nerve fibers were reconnected with endplates (Fig. 5G) and in some cases, several MN axons appear to innervate a single endplate. We weighed the biceps muscle in each group at 8 weeks postinjury. The reinnervated biceps muscle weighed 0.155 ⫾ 0.003 g, whereas the denervated muscle weighed Table 3 Mean soma area in square micrometers (mean ⫾ SEM) of normal and avulsed motoneurons when the PN graft was implanted at 4, 6, or 8 weeks postinjury Weeks

Normal PBS Boc-D-FMK

4

6

8

218.32 ⫾ 9.56 N/A 184.91 ⫾ 8.86

210.47 ⫾ 8.05 N/A 189.61 ⫾ 8.35

217.54 ⫾ 11.74 N/A 199.06 ⫾ 9.79

Ac-DEVD-CHO 44.1 ⫾ 4.8

Boc-D-FMK 52.5 ⫾ 1.65 193.93 ⫾ 5.46 (P ⬎ 0.05) 168.22 ⫾ 2.79 (P ⬎ 0.05)

Ac-DEVD-CHO 0 N/A 129.69 ⫾ 2.53 (P ⬍ 0.001)

only 0.105 ⫾ 0.007 g (P ⬍ 0.001) (Fig. 7). The weight of the reinnervated muscle was similar to normal muscle (0.18 ⫾ 0.005 g; P ⬍ 0.05). These results indicate that the reinnervated muscle does not exhibit significant signs of atrophy. In the intact muscle, postsynaptic AChRs can be localized on the membrane of muscle fibers by labeling with ␣-BTX-TRITC. Normal AChRs were oval in shape with an extensively folded internal structure (Fig. 5D). Four weeks after denervation, the number of AChRs was increased in the biceps and these had an increased surface area and were disorganized. Denervated muscles had a widespread expression of AChRs extrasynaptically (Fig. 5F), whereas 8 weeks after treatment with Boc-D-FMK, the AChRs were organized with less extensive internal foldings and were smaller than normal AChRs (Fig. 5H). These results indicate that neuromuscular junctions in the denervated muscle were reinnervated after treatment with Boc-D-FMK plus a PN bridge. Discussion The present results indicate that caspases play a key role in the death of spinal MNs after injury in neonates. Inhibition of caspases led to long-term neuroprotection as well as axonal regeneration of avulsed spinal MNs. With a PN bridge between the spinal cord and the denervated muscle target, the caspase inhibitor-treated MNs were able to reinnervate the neuromuscular junction and muscular atrophy was reduced. These results suggest that the inhibition of caspases may be a potent strategy for functional recovery following brachial plexus injury. Neurotrophic factors have been suggested as a potential treatment of brachial plexus injury and GDNF is suggested to be the most effective in preventing motoneuron death after root avulsion especially in neonates (Yuan et al., 2000). Viral-mediated delivery of GDNF has been shown to demonstrate longer term neuroprotection of both neonatal and adult motoneurons after injury in rats. In the adult, neuroprotection by adenoviraldelivered GDNF declines from 2 to 8 weeks after root avulsion and the rescue effect is only partial (30%) at 8 weeks postlesion (Watabe et al., 2000). In neonates, 40% of MNs are retained 4 weeks after nerve crush when treated

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Fig. 3. Fluorescent photomicrographs showing FG-labeled MNs after different treatments and at different ages. During normal postnatal development, spinal MNs only express a regenerative capacity when they are older than 2 weeks. Following the administration of caspase inhibitors Boc-D-FMK or Ac-DEVD-CHO; however, MNs could regenerate before this critical period. This suggests that inhibition of caspases may be required for MN regeneration in this situation (see text for details). Scale bar ⫽ 200 ␮m.

with adenoviral-mediated GDNF (Baumgartner and Shine, 1998). However, the same treatment did not rescue neonatal motoneurons from avulsion-induced death. By contrast, we report here that the inhibition of caspases has a prolonged 8-week survival effect on MNs after neonatal root avulsion. After a single dose of Boc-D-FMK, 67% of avulsed MNs remained alive, whereas in the absence of treatment virtually all MNs were dead by 7 days postinjury. Caspase inhibition provides long-term neuroprotection of neonatal spinal MNs after root avulsion. However, since the neuroprotection lasted only for 8 weeks, it is important to investigate whether multiple dosage of caspase inhibitors can prolong the survival rate of injured MNs. GDNF promotes MN regeneration in adult animals fol-

lowing nerve transection (Baumgartner and Shine, 1998; Blesch and Tuszynski, 2001; Chen et al., 2001b) and can activate the MAP kinase and PI 3-kinase signal transduction pathways which are involved in neuronal survival and neurite outgrowth (Sloer et al., 1999; Chen et al., 2001a). In addition, GDNF upregulates the expression of GAP-43 and calcitonin gene-related peptide (CGRP) during the process of axonal regeneration (Blesch and Tuszynski, 2001; Chen et al., 2001a). These studies suggest that GDNF promotes the expression of multiple gene pathways involved in regeneration. However, it is not known whether GDNF is required for the regeneration of neonatal MNs following root avulsion. We report here that treatment with a single dose of a caspase inhibitor promoted significant axonal

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Fig. 4. Photomicrographs showing that caspase inhibitors directly promoted MN regeneration. (A) Regenerative capacity of rat spinal MNs during postnatal development. (B–D) Treatment with either Boc-D-FMK or Ac-DEVD-CHO enhanced the ability of motoneurons to regenerate (***P ⬍ 0.001). Newborn (P1) rats were used to investigate whether caspase inhibitors directly promoted motoneuron regeneration. After the implantation of PN graft and treatment with PBS, Boc-D-FMK, or Ac-DEVD-CHO, animals were allowed to survive for 10 days, which is before the period when MNs normally express a regenerative capacity during postnatal development. MNs of PBS-treated animals failed to regenerate and were FG negative (B). Administration of Boc-D-FMK (C) or Ac-DEVD-CHO (D) promoted MN regeneration resulting in FG-positive cells. Scale bar ⫽ 100 ␮m.

regeneration. Following root avulsion, Boc-D-FMK-treated motoneurons were able to regenerate their axons and reinnervate a target muscle if a PN bridge was provided. Accordingly, caspase inhibition together with a PN bridge may be a potential therapeutic treatment for neonatal brachial plexus injury. The degree of behavioral and electrophysiological recovery by treatment with caspase inhibitor together with PN bridge following brachial plexus injury are currently being examined in our laboratory. We have previously shown that spinal MNs attain a regenerative capacity by the second postnatal week (Chan et al., 2002). In the present study, we found that caspase inhibitors appear to enhance the ability of MNs to regenerate axons. Following caspase inhibition MN regeneration could be demonstrated even in P1 and P7 rats. To investigate whether caspase inhibitors directly promoted axonal regeneration, some P1 animals were allowed to survive for 10 days after the implantation of PN graft, which is a time

point prior to the normal attainment of a regenerative ability. Both Boc-D-FMK and Ac-DEVD-CHO promoted MN regeneration in this situation. These data suggest that caspase inhibitors may be able to directly promote regeneration rather than merely keep MNs alive until they are mature enough to express a regenerative capacity. We are currently examining changes in gene expression following neonatal caspase inhibition in an attempt to determine the mechanism by which regeneration is affected. We find that MNs treated with Boc-D-FMK exhibited regeneration only when the PN graft was either implanted immediately following root avulsion or 2 weeks later, whereas PN implantation at 4, 6, or 8 weeks following root avulsion plus caspase inhibition was ineffective. Although the morphology of the rescued MNs was similar to control contralateral MNs, regeneration did not occur. In addition, Ac-DEVD-CHO could only promote axonal regeneration if the PN graft was implanted immediately after root avulsion.

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Fig. 5. Target muscle reinnervation after treatment with Boc-D-FMK. (A and B) Regenerated motoneurons were labeled by FG injected into the biceps muscle 8 weeks after treatments. In the sham control, no labeled MNs were observed (A), whereas FG-labeled MNs were found on the lesioned side of the spinal cord after treatment with Boc-D-FMK (B). Since FG was injected into the denervated biceps muscle, this indicated that the FG-labeled MNs extend their axons through the PN bridge to reinnervate the biceps. (C, E, and G) Silver and cholinesterase staining showing motor endplates. (C) Bundles of motor endplates were located in the normal biceps muscle with single axons innervating each endplate. (E) By 4 weeks following denervation, only a few short residual axons were observed and there were many nerve-free endplates. (G) Multiple axons innervating single endplates were often found in the biceps muscle 8 weeks after treatment with Boc-D-FMK although nerve-free endplates were also observed. (D, F, and H) Fluorescent photomicrographs showing the morphology of postsynaptic acetylcholine receptor (AChRs) using ␣-bungarotoxin histochemistry. (D) Morphology of normal AChRs. (F) AChRs in the denervated biceps 4 weeks after denervation showing unusual morphology (see text); there were also increasing numbers of disorganized AChRs found in the muscle. (H) By 8 weeks after treatment with Boc-D-FMK, the reinnervated biceps muscle exhibits many morphologically normal AChRs. Scale bar ⫽ 100 ␮m.

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Fig. 6. Photomicrographs showing the organization of the biceps muscles at 8 weeks postinjury. In intact muscles, bundles of fibers are shown in both cross (A) and longitudinal (B) sections. Muscle fibers were evenly distributed (A) and striation of muscles was observed (B). (C and D) Denervated muscles were disorganized with necrotic zones and muscle fibers and striations were abnormal. (E and F) After treatment with Boc-D-FMK, the muscle fibers were more evenly distributed with clearly visible striations. However, fiber size was reduced compared with the normal intact muscle. Scale bar ⫽ 50 ␮m.

These observations suggest that regenerative-promoting genes may only be expressed transiently following injury and therefore that it is important to determine the optimum time point for nerve transplantation. Our data indicate that

Fig. 7. Weights of the biceps muscle after different treatments. The weight of the reinnervated biceps muscle was not significantly different from the normal control muscle.

following neonatal avulsion and caspase inhibition, the first 2 weeks postinjury are optimal for the transplantation of a PN bridge. The PN bridge used here connected the injured MNs to a denervated muscle. In neonates, without treatment with Boc-D-FMK, the injured MNs were unable to extend their axons into the PN bridge so no MNs were labeled by the FG tracer and muscle atrophy occurred. Although a permissive peripheral environment was provided by the PN graft, the injured MNs died rapidly and thus failed to regenerate. In the Boc-D-FMK treated neonates, 39% of motoneurons were labeled by FG 8 weeks after the implantation indicating that MNs had regrown axons into the PN bridge and reinnervated the denervated muscle. Morphologically normal neuromuscular junctions were found in the treated muscle and the weight of muscle was comparable to the normal controls. These results suggest that at least some of the regenerated axons may have reestablished neuromuscular contacts. Since muscle reinnervation is a slow process, we speculate that some regenerating axons had not yet reached

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the target muscle by 8 weeks. It will be important to examine the degree of functional recovery at longer time points by using both electrophysiological and behavioral methods. The PN graft consists of trophic, cell adhesion, and extracellular matrix (ECM) molecules which are essential for axonal regeneration. Schwann cells secrete a mixture of neurotrophic factors, including GDNF, that can rescue CNS neurons from injury (Wu, 1996; Yick et al., 1999). In addition, the graft contains glial cells that produced cell adhesion molecules such as tenascin, neuronal cell adhesion molecules (NCAM), and myelin-associated glycoprotein (MAG) for axonal guidance (Zhang et al., 1995; Dezawa and Nagano, 1996). Components of the ECM such as laminin and fibronectin have been shown to promote regeneration (Wong et al., 1999; Yanagida et al., 1999). Here we show that a PN graft alone is not sufficient to promote MN regeneration in young animals, whereas inhibition of caspases together with the implantation of a PN graft resulted in MN regeneration and reinnervation. This suggests that modifications of both the intrinsic and extrinsic environment are essential for extensive axonal regeneration. This is in line with the recent study showing that treatment with 4-aminopyridine in the neuromuscular junctions improves reinervation following nerve crush in neonates (Dekkers et al., 2001). We have also compared the morphology of neuromuscular junctions in normal, denervated, and reinnervated biceps muscle. Motor endplates were stained by silver and cholinesterase histochemistry. Four weeks after denervation, an increasing number of free (uninnervated) endplates were found. After treatment with Boc-D-FMK, MNs reinnervated the biceps muscle through the PN bridge and morphologically normal endplates in which each endplate was innervated by a single axon were observed. However, in some cases, several axons innervated a single endplate even though some free endplates remained. Normal clusters of AChRs are oval in shape with extensive internal foldings and these AChRs are distributed in a bandlike pattern in the area of the biceps muscle near the musculocutaneous nerve. Four weeks after denervation, an increasing number of AChRs were found in this area. Compared to controls, these AChRs were larger in size and there were less internal foldings. After reinnervation, the AChRs were distributed randomly over the muscle, especially near the implantation site of the PN bridge and they were smaller than normal with some internal foldings. In our model, the C7 MNs were connected to the biceps, a muscle which is normally innervated by the musculocutaneous nerve composed mainly of C5 and C6 derived motor axons. The change in target may account for the distinct distribution pattern of innervation in this situation. We have previously reported that the caspase-3-specific inhibitor Ac-DEVD-CHO rescued more than 80% of the injured spinal MNs when examined 2 weeks after root avulsion (Chan et al., 2001), whereas in the present study the pan-caspase inhibitor Boc-D-FMK demonstrated long-

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term neuroprotection for more than 8 weeks. These results suggest that inhibition of caspase-3 alone may not be sufficient to rescue MNs after brachial plexus injury. However, administration of the pan-caspase inhibitor Boc-D-FMK, which inhibits multiple caspases, was able to promote injured MN regeneration. Because caspase-9, which mediates early stages of apoptosis, is required for the activation of caspase-3 (Li et al., 2001), inhibiting upstream stages in the apoptotic execution pathway may be essential for the rescue of MNs after injury. Therefore, inhibition of caspase-3 alone may not be sufficient for rescuing MNs and promoting regeneration after neonatal avulsion. On the other hand, it is known that some forms of neuronal apoptosis may be caspase-3-independent (Oppenheim et al., 2001). In addition, caspase inhibitors could not rescue all MNs from apoptosis in developing chick embryo (Yaginuma et al., 2001). In a previous study, we showed that the pan-caspase inhibitor Boc-D-FMK or the caspase-3-specific inhibitor Ac-DEVD-CHO could only rescue about 80% of neonatal spinal MN at 7 days after root avulsion (Chan et al., 2001). These data together with the present results suggest that one may need to inhibit both caspase-dependent and caspaseindependent pathways to achieve optimal survival of injured motoneurons.

Conclusion The experiments presented here provide evidence that following root avulsion, neonatal spinal MNs can survive and reinnervate target muscle if appropriate treatment is provided. A single injection of Boc-D-FMK results in longterm protection of MNs against root avulsion-induced death for more than 8 weeks and the Boc-D-FMK-treated MNs are able to regenerate their axons into an implanted PN graft and reinnervate the target muscle. Taken together, these data suggest that local administration of Boc-D-FMK may be a potent treatment for brachial plexus injury in young animals.

Acknowledgments This study was supported by research grants from the University of Hong Kong and the Hong Kong Research Grants Council.

References Allen, J.W., Eldadah, B.A., Huang, X., Knoblach, S.M., Faden, A.I., 2001. Multiple caspases are involved in beta-amyloid-induced neuronal apoptosis. J. Neurosci. Res. 65, 45–53. Bar, J., Dvir, A., Hod, M., Orvieto, R., Merlob, P., Neri, A., 2001. Brachial plexus injury and obstetrical risk factors. Int. J. Gynaecol. Obstet. 73, 21–25.

202

Y.-M. Chan et al. / Experimental Neurology 181 (2003) 190 –203

Barnes, N.Y., Li, L., Yoshikawa, K., Schwartz, L.M., Oppenheim, R.W., Milligan, C.E., 1998. Increased production of amyloid precursor protein provides a substrate for caspase-3 in dying motoneurons. J. Neurosci. 18, 5869 –5880. Baumgartner, B.J., Shine, H.D., 1998. Permanent rescue of lesioned neonatal motoneurons and enhanced axonal regeneration by adenovirusmediated expression of glial cell-line-derived neurotrophic factor. J. Neurosci. Res. 54, 766 –777. Blesch, A., Tuszynski, M.H., 2001. GDNF gene delivery to injured adult CNS motor neurons promotes axonal growth, expression of the trophic neuropeptide CGRP, and cellular protection. J. Comp. Neurol. 436, 399 – 410. Blondet, B., Ait-Ikhlef, A., Murawsky, M., Rieger, F., 2001. Transient massive DNA fragmentation in nervous system during the early course of a murine neurodegenerative disease. Neurosci. Lett. 305, 202–206. Bunge, R.P., 1994. The role of the Schwann cell in trophic support and regeneration. J. Neurol. 242, S19 –21. Carbonetto, S., 1991. Facilitatory and inhibitory effects of glial cells and extracellular matrix in axonal regeneration. Curr. Opin. Neurobiol. 1, 407– 413. Carlstedt, T., Anand, P., Hallin, R., Misra, P.V., Noren, G., Seferlis, T., 2000. Spinal nerve root repair and reimplantation of avulsed ventral roots into the spinal cord after brachial plexus injury. J. Neurosurg. 93, 237–247. Chai, H., Wu, W., So, K.F., Yip, H.K., 2000. Survival and regeneration of motoneurons in adult rats by reimplantation of ventral root following spinal root avulsion. NeuroReport 11, 1249 –1252. Chan, Y.M., Wu, W., Yip, H.K., So, K.F., 2002. Development of the regenerative capacity of postnatal axotomized rat spinal motoneurons. NeuroReport 13, 1071–1074. Chan, Y.M., Wu, W., Yip, H.K., So, K.F., Oppenheim, R.W., 2001. Caspase inhibitors promote the survival of avulsed spinal motoneurons in neonatal rats. NeuroReport 12, 541–545. Chen, Z., Chai, Y., Cao, L., Huang, R., Cui, R., Lu, C., He, C., 2001a. Glial cell line-derived neurotrophic factor promotes survival and induces differentiation through the phosphatidylinositol 3-kinase and mitogenactivated protein kinase pathway respectively in PC12 cells. Neuroscience 104, 593–598. Chen, Z., Chai, Y.F., Cao, L., Lu, C.L., He, C., 2001b. Glial cell linederived neurotrophic factor enhances axonal regeneration following sciatic nerve transection in adult rats. Brain Res. 902, 272–276. Clarke, P.G., Oppenheim, R.W., 1995. Neuron death in vertebrate development: in vitro methods. Methods Cell. Biol. 46, 277–321. David, S., Aguayo, A.J., 1981. Axonal elongation into peripheral nervous system “bridges” after central nervous system injury in adult rats. Science 214, 931–933. David, S., Aguayo, A.J., 1985. Axonal regeneration after crush injury of rat central nervous system fibres innervating peripheral nerve grafts. J. Neurocytol. 14, 1–12. Dekkers, J., Waters, J., Vrbova, G., Greensmith, L., 2001. Treatment of the neuromuscular junction with 4-aminopyridine results in improved reinnervation following nerve injury in neonatal rats. Neuroscience 103, 267–274. Dezawa, M., Nagano, T., 1996. Immunohistochemical localization of cell adhesion molecules and cell-cell contact proteins during regeneration of the rat optic nerve induced by sciatic nerve autotransplantation. Anat. Rec. 246, 114 –126. Emery, E., Rhrich-Haddout, F., Kassar-Duchossoy, L., Lyoussi, B., Tadie, M., Horvat, J.C., 2000. Motoneurons of the adult marmoset can grow axons and reform motor endplates through a peripheral nerve bridge joining the locally injured cervical spinal cord to the denervated biceps brachii muscle. J. Neurosci. Res. 62, 821– 829. Emery, E., Horvat, J.C., Tadie, M., 1997. Motor reconnection between the damaged cervical cord and the denervated biceps muscle using an autologous peripheral nerve segment in the adult marmoset. Chirurgie 122, 252–259.

Fournier, H.D., Menei, P., Khalifa, R., Mercier, P., 2001. Ideal intraspinal implantation site for the repair of ventral root avulsion after brachial plexus injury in humans: a preliminary anatomical study. Surg. Radiol. Anat. 23, 191–195. Gerhardt, E., Kugler, S., Leist, M., Beier, C., Berliocchi, L., Volbracht, C., Weller, M., Bahr, M., Nicotera, P., Schulz, J.B., 2001. Cascade of caspase activation in potassium-deprived cerebellar granule neurons: targets for treatment with peptide and protein inhibitors of apoptosis. Mol. Cell. Neurosci. 17, 717–731. Guenard, V., Kleitman, N., Morrissey, T.K., Bunge, R.P., Aebischer, P., 1992. Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration. J. Neurosci. 12, 3310 –3320. Hayashi, T., Sakurai, M., Abe, K., Sadahiro, M., Tabayashi, K., Itoyama, Y., 1998. Apoptosis of motor neurons with induction of caspases in the spinal cord after ischemia. Stroke 29, 1007–1012. Hayashi, Y., Jikihara, I., Yagi, T., Fukumura, M., Ohashi, Y., Ohta, Y., Takagi, H., Maeda, M., 2001. Immunohistochemical investigation of caspase-1 and effect of caspase-1 inhibitor in delayed neuronal death after transient cerebral ischemia. Brain Res. 893, 113–120. Hoeksma, A.F., Wolf, H., Oei, S.L., 2000. Obstetrical brachial plexus injuries: incidence, natural course and shoulder contracture. Clin. Rehabil. 14, 523–526. Hopkins, W.G., Slack, J.R., 1981. The sequential development of nodal sprouts in mouse muscles in response to nerve degeneration. J. Neurocytol. 10, 537–556. Horvat, J.C., 1991. Transplants of fetal neural tissue and autologous peripheral nerves in an attempt to repair spinal cord injuries in the adult rat. An overall view. Paraplegia 29, 299 –308. Horvat, J.C., Pecot-Dechavassine, M., Mira, J.C., Davarpanah, Y., 1989. Formation of functional endplates by spinal axons regenerating through a peripheral nerve graft: a study in the adult rat. Brain Res. Bull. 22, 103–114. Jamieson, A., Hughes, S., 1980. The role of surgery in the management of closed injuries to the brachial plexus. Clin. Orthop. 147, 210 –215. Li, L., Oppenheim, R.W., Milligan, C.E., 2001. Characterization of the execution pathway of developing motoneurons deprived of trophic support. J. Neurobiol. 46, 249 –264. Li, L., Houenou, L.J., Wu, W., Lei, M., Prevette, D.M., Oppenheim, R.W., 1998. Characterization of spinal motoneuron degeneration following different types of peripheral nerve injury in neonatal and adult mice. J. Comp. Neurol. 396, 158 –168. Liu, Z., Martin, L.J., 2001a. Isolation of mature spinal motor neurons and single-cell analysis using the comet assay of early low-level DNA damage induced in vitro and in vivo. J. Histochem. Cytochem. 49, 957–972. Liu, Z., Martin, L.J., 2001b. Motor neurons rapidly accumulate DNA single-strand breaks after in vitro exposure to nitric oxide and peroxynitrite and in vivo axotomy. J. Comp. Neurol. 432, 35– 60. Lo, A.C., Houenou, L.J., Oppenheim, R.W., 1995. Apoptosis in the nervous system: morphological features, methods, pathology, and prevention. Arch. Histol. Cytol. 58, 139 –149. Martin, L.J., 1999. Neuronal death in amyotrophic lateral sclerosis is apoptosis: possible contribution of a programmed cell death mechanism. J. Neuropathol. Exp. Neurol. 58, 459 – 471. Milligan, C.E., Prevette, D., Yaginuma, H., Homma, S., Cardwell, C., Fritz, L.C., Tomaselli, K.J., Oppenheim, R.W., Schwartz, L.M., 1995. Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro. Neuron 15, 385–393. Morrissey, T.K., Kleitman, N., Bunge, R.P., 1991. Isolation and functional characterization of Schwann cells derived from adult peripheral nerve. J. Neurosci. 11, 2433–2442. Noetzel, M.J., Park, T.S., Robinson, S., Kaufman, B., 2001. Prospective study of recovery following neonatal brachial plexus injury. J. Child Neurol. 16, 488 – 492. Oliveira, A.L., Risling, M., Deckner, M., Lindholm, T., Langone, F., Cullheim, S., 1997. Neonatal sciatic nerve transection induces TUNEL

Y.-M. Chan et al. / Experimental Neurology 181 (2003) 190 –203 labeling of neurons in the rat spinal cord and DRG. NeuroReport 8, 2837–2840. Oppenheim, R.W., Flavell, R.A., Vinsant, S., Prevette, D., Kuan, C.Y., Rakic, P., 2001. Programmed cell death of developing mammalian neurons after genetic deletion of caspases. J. Neurosci. 21, 4752– 4760. Oppenheim, R.W., Cole, T., Prevette, D., 1989. Early regional variations in motoneuron numbers arise by differential proliferation in the chick embryo spinal cord. Dev. Biol. 133, 468 – 474. Pecot-Dechavassine, M., Mira, J.C., 1994. Electrophysiological evaluation of the effects of naftidrofuryl on skeletal muscle reinnervation in the rat. Microsurgery 15, 116 –122. Rhrich-Haddout, F., Kassar-Duchossoy, L., Bauchet, L., Destombes, J., Thiesson, D., Butler-Browne, G., Lyoussi, B., Baillet-Derbin, C., Horvat, J.C., 2001. Alpha-motoneurons of the injured cervical spinal cord of the adult rat can reinnervate the biceps brachii muscle by regenerating axons through peripheral nerve bridges: combined ultrastructural and retrograde axonal tracing study. J. Neurosci. Res. 64, 476 – 486. Richardson, P.M., Issa, V.M., Aguayo, A.J., 1984. Regeneration of long spinal axons in the rat. J. Neurocytol. 13, 165–182. Richardson, P.M., Issa, V.M., Shemie, S., 1982. Regeneration and retrogradedegeneration of axons in the rat optic nerve. J. Neurocytol. 11, 949 –966. Rothstein, J.D., Bristol, L.A., Hosler, B., Brown Jr., R.H., Kuncl, R.W., 1994. Chronic inhibition of superoxide dismutase produces apoptotic death of spinal neurons. Proc. Natl. Acad. Sci. USA 91, 4155– 4159. Sloer, R.M., Dolcet, X., Encinas, M., Egea, J., Bayascas, J.R., Comella, J.X., 1999. Receptors of the glial cell line-derived neurotrophic factor family of neurotrophic factors signal cell survival through the phosphatidylinositol 3-kinase pathway in spinal cord motoneurons. J. Neurosci. 19, 9160 –9169. Sorbie, C., Porter, T.L., 1969. Reinnervation of paralysed muscles by direct motor nerve implantation: an experimental study in the dog. J. Bone Joint Surg. Br. 51, 156 –164. Turgeon, V.L., Lloyd, E.D., Wang, S., Festoff, B.W., Houenou, L.J., 1998. Thrombin perturbs neurite outgrowth and induces apoptotic cell death in enriched chick spinal motoneuron cultures through caspase activation. J. Neurosci. 18, 6882– 6891. Villegas-Perez, M.P., Vidal-Sanz, M., Bray, G.M., Aguayo, A.J., 1988. Influences of peripheral nerve grafts on the survival and regrowth of axotomized retinal ganglion cells in adult rats. J. Neurosci. 8, 265–280. Von Coelln, R., Kugler, S., Bahr, M., Weller, M., Dichgans, J., Schulz, J.B., 2001. Rescue from death but not from functional impairment: caspase inhibition protects dopaminergic cells against 6-hydroxydopamine-induced apoptosis but not against the loss of their terminals. J. Neurochem. 77, 263–273.

203

Watabe, K., Ohashi, T., Sakamoto, T., Kawazoe, Y., Takeshima, T., Oyanagi, K., Inoue, K., Eto, Y., Kim, S.U., 2000. Rescue of lesioned adult rat spinal motoneurons by adenoviral gene transfer of glial cell linederived neurotrophic factor. J. Neurosci. Res. 60, 511–519. Wong, K.C., Meyer, T., Harding, D.I., Dick, J.R., Vrbova, G., Greensmith, L., 1999. Integrins at the neuromuscular junction are important for motoneuron survival. Eur. J. Neurosci. 11, 3287–3292. Wu, W., 1993. Expression of nitric-oxide synthase (NOS) in injured CNS neurons as shown by NADPH diaphorase histochemistry. Exp. Neurol. 120, 153–159. Wu, W., 1996. Potential roles of gene expression change in adult rat spinal motoneurons following axonal injury: a comparison among c-jun, offaffinity nerve growth factor receptor (LNGFR), and nitric oxide synthase (NOS). Exp. Neurol. 141, 190 –200. Wu, W., Li, L., 1993. Inhibition of nitric oxide synthase reduces motoneuron death due to spinal root avulsion. Neurosci. Lett. 153, 121–124. Wu, W., Han, K., Li, L., Schinco, F.P., 1994. Implantation of PNS graft inhibits the induction of neuronal nitric oxide synthase and enhances the survival of spinal motoneurons following root avulsion. Exp. Neurol. 129, 335–339. Yaginuma, H., Shiraiwa, N., Shimada, T., Nishiyama, K., Hong, J., Wang, S., Momoi, T., Uchiyama, Y., Oppenheim, R.W., 2001. Caspase activity is involved in, but is dispensable for, early motoneuron death in the chick embryo cervical spinal cord. Mol. Cell. Neurosci. 18, 168 – 182. Yanagida, H., Tanaka, J., Maruo, S., 1999. Immunocytochemical localization of a cell adhesion molecule, integrin alpha5beta1, in nerve growth cones. J. Orthop. Sci. 4, 353–360. Yick, L.W., Wu, W., So, K.F., Yip, H.K., 1999. Peripheral nerve graft and neurotrophic factors enhance neuronal survival and expression of nitric oxide synthase in Clarke’s nucleus after hemisection of the spinal cord in adult rat. Exp. Neurol. 159, 131–138. Yoshiyama, Y., Yamada, T., Asanuma, K., Asahi, T., 1994. Apoptosis related antigen, Le(Y) and nick-end labeling are positive in spinal motor neurons in amyotrophic lateral sclerosis. Acta Neuropathol. (Berl.) 88, 207–211. Yuan, Q., Wu, W., So, K.F., Cheung, A.L., Prevette, D.M., Oppenheim, R.W., 2000. Effects of neurotrophic factors on motoneuron survival following axonal injury in newborn rats. NeuroReport 11, 2237–2241. Zhang, Y., Campbell, G., Anderson, P.N., Martini, R., Schachner, M., Lieberman, A.R., 1995. Molecular basis of interactions between regenerating adult rat thalamic axons and Schwann cells in peripheral nerve grafts I. Neural cell adhesion molecules. J. Comp. Neurol. 361, 193– 209.